MicroRNAs (miRNAs) are indispensable regulators of gene expression that are required for animal development and physiology. In addition, miRNAs have been implicated in a wide spectrum of human diseases, notably cardiovascular disease, neurodegenerative disease, diabetes, and cancer. However, the biological functions of only a small number of individual miRNAs have been described. The identification of pathways and processes directly regulated by individual miRNAs and the identification of specific miRNA targets is vital to understand their role in animal development as well as in human disease. As an eminently genetically tractable animal, C. elegans provides an ideal model system in which to study the functions of miRNAs, particularly as many miRNAs show complete or near-complete conservation between worms and humans. In C. elegans, most miRNAs are not individually required for development;worms carrying mutations in miRNA genes develop essentially normally. It is likely that miRNAs function with other miRNAs and with additional regulatory factors to control developmental pathways. To reveal such complex interactions, we have examined the effects of mutations in individual miRNAs in genetically sensitized backgrounds. We have identified miRNA- dependent phenotypes in a genetic background with lower overall miRNA activity due to loss of an Argonaute-encoding gene, alg-1. In this proposal, we will analyze a single miRNA-dependent phenotype that is associated with the loss of five miRNAs. The objective of this proposal is to identify the biological pathways and direct mRNA targets regulated by these five suppressor miRNAs. The specific aims in this proposal are to: 1) define the genetic pathway in which suppressor miRNAs function. To do this, we will perform genetic analysis to test for interactions with developmental timing genes, miRNA pathway genes, and individual miRNA genes, for which a function has been described and 2) characterize the molecular mechanism for miRNA-dependent suppression of alg-1 developmental timing defects. To do this, we will determine if suppressor miRNAs affect the biogenesis or activity of miRNAs, examine candidate miRNA targets, and perform transcriptome analysis to identify the profile of misregulated genes in worms missing suppressor miRNA activity. PUBLIC HEALTH RELEVANCE: This proposal focuses on miRNAs conserved between worms and mammals. Achieving the goals of this project will provide key information about pathways regulated by miRNAs during development that may be misregulated in human disease and to further describe the biological principles of miRNA regulation of target mRNAs in animals. PUBLIC HEALTH RELEVANCE: MicroRNAs are indispensable regulators of gene expression that are required for animal development and physiology. In addition, microRNAs have been implicated in a wide spectrum of human diseases, notably cardiovascular disease, neurodegenerative disease, diabetes, and cancer. It is not clear whether changes in microRNA activity are a cause or a consequence of human disease. The identification of biological functions of individual microRNAs is vital to understand their role in animal development as well as in human disease.